The present invention is generally related to polynucleotide molecules and to their use for production of desired products after introduction thereof into human or animal cells.
More specifically, the present invention is directed to alphavirus expression vectors comprising at least part of an alphavirus genome and heterologous RNA inserted downstream of an alphavirus base sequence having translation enhancing activity. Such vectors can be used to achieve enhanced levels of expression of DNA or cDNA coding for a desired product and being complementary to said heterologous RNA after introduction of said vector in eukaryotic cells in cell culture or in a living body.
The modern techniques in molecular biology have opened up many completely unforeseen possibilities for the pharmaceutical and biotechnological industry. For instance, it is today possible to clone any gene (once identified) as a DNA (or cDNA) molecule and express it in prokaryotic and/or eukaryotic cells. This has facilitated the production of the corresponding proteins in vitro for therapeutic use and/or prophylactic use, e.g. as a vaccine. Lately the possibilities of gene expression technology have been extended also to in vivo use in whole animals and even in human beings. Illustrative of this recent development are human gene therapy .sup.(1) and genetic immunization .sup.(2). In order to express a foreign gene in cells in vivo or in vitro, the gene segment, or the corresponding cDNA, comprising the coding sequences, is usually inserted into a so called expression vector. This vector provides for all elements that are necessary for the transcription and translation of the gene, or the cDNA thereof, within the cell. Today, there exist very efficient expression vectors for bacterial and yeast cells. However, this is not the case for animal, such as mammalian, cells. This creates a large problem when a protein with mammalian specific-modifications has to be produced and isolated for a therapeutic purpose or for prevention of disease or in cases where expression in whole organisms, e.g. the living body of an animal or human, is required. Therefore, in very general terms there is a great demand for new expression vectors for use in animal, e.g. mammalian, cells, which vectors have
(i) increased protein expression efficiency, PA1 (ii) broadened host cell specificity, and PA1 (iii) increased safety.
Recently, a completely new type of DNA expression vectors have been developed for use in animal, e.g. mammalian, cells. These vectors are based on the alphavirus genome.
Alphavirus is a genus belonging to the family Togaviridae having single stranded RNA genomes of positive polarity enclosed in a nucleocapsid surrounded by an envelope containing viral spike proteins.
The Alphavirus genus comprises among others the Sindbis virus, the Semliki Forest virus (SFV), the Ross River virus and Venezuelan, Western and Eastern equine encephalitis viruses, which are all closely related. In particular, the Sindbis and the Semliki Forest viruses have been widely studied and the life cycle, mode of replication, etc, of these viruses are well known and thus, need not to be specifically discussed herein.
Alphaviruses replicate very efficiently in animal cells which makes them valuable as vectors for production of protein and nucleic acids in such cells.
Expression systems based on the Sindbis virus are disclosed in U.S. Pat. No. 5,091,309 and U.S. Pat. No. 5,217,879. The Sindbis virus vectors of U.S. Pat. No. 5,091,309 comprise RNA derived from Sindbis defective interfering (DI) RNA having heterologous RNA inserted therein. In U.S. Pat. No. 5,217,879 self-replicating and self-packaging recombinant Sindbis virus RNA molecules are disclosed comprising a heterologous coding sequence and at least one Sindbis virus junction region able to direct Sindbis virus subgenomic messenger RNA synthesis in a host cell. RNA transcripts are synthesized in vitro by transcription of Sindbis virus cDNA which has been inserted in a plasmid under control of a promoter, such as SP6.
Xiong et al., Science, Vol 243, 1989, 1188-1191 .sup.(3) also disclose a gene expression system based on Sindbis virus. This system is said to be efficient in a broad range of animal cells. Expression of the bacterial CAT (chloramphenicol acetyltransferase) gene in insect, avian and mammalian cells inclusive of human cells is disclosed therein.
In Bio/Technology, Volume 9, pages 1356-1361, 1991 .sup.(4), Liljestrom and Garoff disclose animal cell expression vectors based on the SFV replicon. When foreign DNA coding sequences are inserted into these vectors, high amounts of foreign protein are obtained.
According to WO 92/10578, an RNA molecule is provided, which is derived from an alphavirus RNA genome and is capable of efficient infection of animal cells, which RNA molecule comprises the complete alphavirus genome regions, which are essential for replication of the said alphavirus RNA, and further comprises an exogenous RNA sequence capable of expressing its function in said host cell, said exogenous RNA sequence being inserted into a region of the RNA molecule which is non-essential to replication thereof. According to WO 92/10578 such RNA molecules can be transferred into animal cells by any means of transfection or by packaging of said RNA molecules into infectious alphavirus particles for later infection of animal cells. In both cases the transfected or infected RNA molecule will be able to replicate within the target animal cell and to express the exogenous RNA sequences inserted into said RNA molecule. Such molecules and strategies for their expression within the cell can be used as vaccines or strategies to vaccinate in order to prevent or treat infection or cancer. In this reference, SFV has been used to illustrate alphaviruses.
The above mentioned expression vectors based on the alphavirus genome have been shown to promote a higher protein expression efficiency than earlier mammalian protein expression systems. They have also been shown to work in almost all higher eukaryotic cell types. Furthermore, they have been complemented with highly stringent safety features to prevent spreading of the virus .sup.(5). Important proteins, to be used for prevention of disease, like the HIV spike protein, have been produced with this system and such proteins have been shown to have a more native-like structure than when produced in other systems .sup.(6). Alphavirus vectors have also been used successfully for genetic immunization .sup.(7).